Introduction: Despite half a century of research on vitamin D (Vit-D), its link to ethanol (Eth.) abuse and dependence has only been discussed in recent decades. Evidence also shows the involvement of Vit-D in the development of dopaminergic neurons in the nucleus accumbens, an increase in the expression of tyrosine hydroxylase, and the regulation of dopaminergic processes. Our novel idea for this work was based on the effectiveness of Vit-D on the dopamine signaling pathway. Methods and Materials:In this study, male Wistar rats 200-250 gr were used. The rats (n=42) were assigned into six equal groups (control [saline gavage equivalent to the drug], vehicle [sessom oil, i.p. 30 min before the Eth. gavage], Eth. [Eth., 1.25 mg/kg/ gavage for 8 days], and Vit-D [250, 500, and 1000 IU/kg, i.p.]). Prior to each conditioning session using a ten-day conditioned place preference protocol, the animals received Vit-D injection. Afterward, the locomotor activity of the animals was assessed using open-field apparatus. Conditioning preference index (sec.) was recorded as a criterion for Eth. preference. One-way analysis of variance (ANOVA) and post-hoc Tukey’s test were used for statistical analyses. Results: Non-parametric ANOVA revealed that the conditioning preference index was increased in both Eth. and vehicle groups (p<0.001 for both groups); however post-hoc Tukey’s test revealed that Vit.-D administration before Eth. (250 and 500 mg/kg), caused a significant decrease (p<0.05 and p<0.001, respectively) in the place preference index in the acquisition phase, compared to the vehicle group. No significant difference was seen in the locomotor activity of the animals (p>0.05). Conclusion: Thepre-treatment Vit.-D interacts via dopamine signaling to attenuate the Eth. effect. It is therefore presumed that Vit-D can be considered an effective therapeutic approach for ethanol addiction and abuse.